FIGURE 4.
Asna-1 and WRB are involved in the posttranslational ER membrane insertion of BNLF2a. In vitro translation reactions were performed in wheat germ extract in the presence of [35S]Met using truncated BNLF2aC8-NST mRNA templates lacking a stop codon. After translation, BNLF2aC8-NST containing ribosome-nascent chain complexes were collected by centrifugation through a sucrose cushion and resuspended in microsomal membrane containing rabbit reticulocyte lysate in the presence of either 200 μg/ml R9L peptide as mock control (-) or WRB-67 inhibitory peptide (A, 200 μg/ml; B, concentrations as indicated). After ribosomal release by puromycin and RNase treatment, samples were further incubated for 30 min at 32 °C. Translation products were analyzed by Tricine/SDS-PAGE (10%) and visualized by phosphoimaging. Histograms show the average amount of glycosylated BNLF2a protein. The error bars indicate the mean ± S.D. of three independent experiments. *, p = 0.05 (Student's t test).